title: Living colors in the gray mold pathogen Botrytis cinerea: codon-optimized genes encoding green fluorescent protein and mCherry, which exhibit bright fluorescence.
abstract: The green fluorescent protein (GFP) and its variants have been widely used in modern biology as reporters that allow a variety of live-cell imaging techniques. So far, GFP has rarely been used in the gray mold fungus Botrytis cinerea because of low fluorescence intensity. The codon usage of B. cinerea genes strongly deviates from that of commonly used GFP-encoding genes and reveals a lower GC content than other fungi. In this study, we report the development and use of a codon-optimized version of the B. cinerea enhanced GFP (eGFP)-encoding gene (Bcgfp) for improved expression in B. cinerea. Both the codon optimization and, to a smaller extent, the insertion of an intron resulted in higher mRNA levels and increased fluorescence. Bcgfp was used for localization of nuclei in germinating spores and for visualizing host penetration. We further demonstrate the use of promoter-Bcgfp fusions for quantitative evaluation of various toxic compounds as inducers of the atrB gene encoding an ABC-type drug efflux transporter of B. cinerea. In addition, a codon-optimized mCherry-encoding gene was constructed which yielded bright red fluorescence in B. cinerea.
authors.affiliation: Department of Biology, University of Kaiserslautern, Kaiserslautern, Germany. mleroch@rhrk.uni-kl.de
authors.forename: Michaela
authors.initials: M
authors.lastname: Leroch
authors.forename: Dennis
authors.initials: D
authors.lastname: Mernke
authors.forename: Dieter
authors.initials: D
authors.lastname: Koppenhoefer
authors.forename: Prisca
authors.initials: P
authors.lastname: Schneider
authors.forename: Andreas
authors.initials: A
authors.lastname: Mosbach
authors.forename: Gunther
authors.initials: G
authors.lastname: Doehlemann
authors.forename: Matthias
authors.initials: M
authors.lastname: Hahn
mesh_terms: D020171:Botrytis / Q000235:genetics / Q000378:metabolism*; D003062:Codon; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling / Q000379:methods; D000097573:Red Fluorescent Protein
chemical_list: D003062:Codon; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein